Lipotropes (methyl group containing nutrients including choline, methionine, folic acid, and vitamin B12) have been shown to have oncostatic action on mammary cancer. We hypothesize that excess lipotropes may alter expression of apoptosis-related genes including bcl-2 gene, via alterations in DNA methylation, and consequently increase the sensitivity of cancer cells to programmed cell death. Specific aims of the proposed study are: 1) to confirm if excess lipotropes increase the susceptibility of breast cancer cells to apoptosis, 2) to investigate if lipotrope-supplementation alters the expression of certain genes involved in the regulation of apoptosis, and 3) to examine if an excess of lipotropes affects genomic methylation patterns of apoptosis-related genes. Tamoxifen (TAM), an anti-cancer agent, will be used to induce apoptosis in breast cancer cells. Two breast cancer cell lines, MCF-7 and T47D, as well as a normal mammary cell line, MCF-10A, will be tested in this study. Cells will be cultured in preincubation medium until 80 percent confluent and then switched to apoptosis induction media (TAM added) with (treatment) or without (control) excess lipotropes. Apoptosis will be accessed by immunohistochemistry, electrophoretic DNA fragmentation patterns, and caspase assay. Expression of apoptosis-related genes will be elucidated by gene array methodology and Northern analysis. The genomic DNA methylation patterns of apoptosis-related genes will be analyzed by methylation specific PCR and HPLC. Direct molecular and biochemical information on the possible effect of excess lipotropes upon breast cancer cell death could ultimately lead to the development of dietary compounds and chemotherapeutic agents that would reduce and treat breast cancer.